Streptococcus uberis adhesion molecule

ABSTRACT

A polypeptide, designated as “ Streptococcus uberis  Adhesion Molecule” (SUAM), and fragments of SUAM, prevent internalization and adherence of  Streptococcus uberis  and other streptococcal pathogens to cells. The SUAM polypeptide and fragments may be used diagnostically and therapeutically. Nucleic acid sequences encoding the SUAM polypeptide and fragments are included in the invention.

[0001] This application claims the priority of pending U.S. ProvisionalPatent Application Ser. No. 60/429,499, filed on Nov. 26, 2002, whichapplication is incorporated herein by reference.

[0002] The invention was developed in part by a research grant from theUnited States Department of Agriculture and the U.S. government maytherefore have certain rights to the invention.

FIELD OF THE INVENTION

[0003] The invention pertains generally to the field of antigenicproteins and polypeptides. Specifically, the invention pertains to thefield of polypeptides that are useful to diagnose the presence of aninfection and to elicit an immune response against a bacterial pathogen,especially streptococcal pathogens.

BACKGROUND OF THE INVENTION

[0004]Streptococcus is a genus of bacteria that causes disease in humansand other animals. In humans, one of the most important streptococcalpathogens is Streptococcus pyogenes, the causative organism of strepthroat, scarlet fever, and rheumatic fever. In cattle, streptococcalinfections are a significant cause of disease, such as mastitis.

[0005] Mastitis affects virtually every dairy farm and has beenestimated to affect 38% of all cows. The disease causes destruction ofmilk-synthesizing tissues which reduces milk production and alters milkcomposition. In severe cases, the productive performance of dairy cattlemay be diminished permanently. Thus, mastitis continues to be the singlegreatest impediment to profitable dairy production. Losses associatedwith mastitis cost American dairy producers about 2 billion dollars peryear and cost dairy producers worldwide an estimated 25 billion dollarsper year.

[0006] Current mastitis control programs devised in the 1960's are basedprimarily on hygiene including teat disinfection, antibiotic therapy andculling of chronically infected cows. Acceptance and application ofthese measures has led to considerable progress in controllingcontagious mastitis pathogens such as Streptococcus agalactiae andStaphylococcus aureus. However, postmilking teat disinfection andantibiotic dry cow therapy have been less effective againstenvironmental mastitis pathogens. Studies have shown that as theprevalence of contagious mastitis pathogens was reduced, the proportionof intramammary infections (IMI) by environmental pathogens increasedmarkedly.

[0007] Therefore, it is not surprising that environmental mastitis hasbecome a major problem in many well-managed dairy farms that havesuccessfully controlled contagious pathogens. In these herds,environmental streptococci account for a significant number of bothsubclinical and clinical IMI in lactating and nonlactating cows.Environmental Streptococcus species involved in bovine mastitis includeStreptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae,Streptococcus equinus (formerly referred to as Streptococcus bovis),Streptococcus equi, Streptococcus parauberis and Streptococcus canis.Among the environmental streptococci, S. uberis and S. dysgalactiaesubsp. dysgalactiae appear to be the most prevalent, infecting mammaryglands as favorable conditions arise.

[0008] In spite of the economic impact caused by the high prevalence ofenvironmental streptococci in many well-managed dairy herds, virulencefactors associated with pathogenesis of environmental streptococcalmastitis in dairy cows are not well understood. This constitutes a majorobstacle for development of strategies to control these importantmastitis pathogens. Consequently, strategies for controlling mastitiscaused by environmental streptococci are poorly defined and currentlyinadequate.

[0009] A significant need exists for effective therapies to combatstreptococcal infections, both in domestic animals and in people, andfor effective modalities by which the presence of a streptococcalinfection may be definitively diagnosed.

[0010] Survival of pathogenic microorganisms, such as Streptococci, hasdepended on the evolution of a range of strategies for evasion of hostdefenses. Associated with this evolution is the expression of a varietyof virulence determinants that favor persistence of bacteria in the faceof a massive inflammatory cell infiltration. In the case of bovinemastitis, it is hypothesized that adherence to and subsequentinternalization of mastitis pathogens into mammary epithelial cells isan important early event in the establishment of new intramammaryinfections in lactating and nonlactating mammary glands of dairy cows.Virulence factors that favor adherence and internalization to host cellsplay a crucial role in the establishment, spread, and persistence ofinfection. During the last decade, research from our laboratory hasfocused extensively on development of in vivo and in vitro models tostudy host-pathogen interactions, and especially on identification andcharacterization of virulence factors associated with the pathogenesisof S. uberis mastitis.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 is a series of bar graphs showing the effects of antibodiesdirected against SUAM (A and B) and pepSUAM (C and D) on adherence andinternalization of S. uberis into bovine mammary epithelial cells.

[0012]FIG. 2 is a diagrammatic representation of a proposed lactoferrinbridge model for adherence of Streptococcus uberis to bovine mammaryepithelial cells.

[0013]FIG. 3 is the theoretically elucidated SUAM gene sequence. (Seq.ID No. 1)

[0014]FIG. 4 shows the translation of the nucleotide sequence of Seq. IDNo. 1 in the correct reading frame. (Seq. ID No. 2)

[0015]FIG. 5 is the DNA sequence of the SUAM gene. (Seq. ID No. 3)

[0016]FIG. 6 shows the translation of the nucleotide sequence of Seq. IDNo. 3 in the correct reading frame. (Seq. ID No. 13 to Seq. No. 17)

DESCRIPTION OF THE INVENTION

[0017] In this application, the terms “Streptococcus uberis AdhesionMolecule” or “SUAM” is preferably used although the terms “StreptococcusLactoferrin-binding Protein”, “Lactoferrin Binding Protein” and “LBP”are also used to refer to the same polypeptide. The terms “Streptococcusuberis Adhesion Molecule” and “SUAM” are preferred so as not to confusethe polypeptide of the present invention with the protein identified as“Streptococcus uberis Lactoferrin-Binding Protein” in Jiang et al., WO98/21231. The Jiang protein is a different protein than the SUAM of thepresent invention. Protein-nucleic acid TBLASTN (National Center forBiotechnology Information) and Swissprot amino acid data bank were usedto align the SUAM N-terminal amino acid sequence with previouslysequenced genes and proteins including S. uberis LBP described by Jianget al. No similarities were found, thus indicating that the SUAMbacterial protein of the invention is novel.

[0018] Recently, it has been shown that S. uberis binds to purifiedbovine milk lactoferrin (LF) and that at least two proteins from S.uberis were involved in this binding. Fang and Oliver, FEMS Microbiol.Lett., 176:91 (1999). It has further been shown that LF appears tofunction as a bridging molecule between S. uberis and bovine mammaryepithelial cells, facilitating adherence of this mastitis pathogen tohost cells. Fang, et al., American Journal of Veterinary Research,61:275 (2000). This research indicates that the S. uberis proteins thatbind to LF influence adherence of S. uberis to mammary epithelial cellsand internalization of S. uberis into bovine mammary epithelial cells.

[0019] Further research in our laboratory has provided the followingdiscoveries.

[0020] (1) A 112 kDA protein from S. uberis that binds to LF wasisolated and purified and an N-terminal amino acid sequence of this 112kDa protein was determined. The sequence is that of a novel protein,which is referred to herein as Streptococcus uberis Adhesion Molecule orSUAM.

[0021] (2) SUAM-like proteins were identified in other Streptococci,including Streptococcus dysgalactiae subsp. dysgalactiae andStreptococcus agalactiae.

[0022] (3) The SUAM-like proteins produced by S. dysgalactiae subsp.dysgalactiae bind to bovine LF in a manner similar to that which occurswith S. uberis.

[0023] (4) Antibodies against SUAM (whole protein) and to a syntheticpeptide (pepSUAM) encompassing 15 amino acids near the N-terminus ofSUAM have been produced.

[0024] (5) These antibodies cross-react with homologous proteins presentin other strains of S. uberis demonstrating that SUAM was produced byall strains of S. uberis evaluated.

[0025] (6) Anti-pepSUAM and anti-SUAM antibodies cross-react with otherstreptococcal pathogens, including S. agalactiae, S. dysgalactiae subsp.dysgalactiae, and Streptococcus pyogenes.

[0026] (7) Antibodies directed against pepSUAM or SUAM inhibit adherenceof S. uberis to, and internalization of S. uberis into, bovine mammaryepithelial cells. This establishes that pepSUAM and SUAM arebiologically active and are involved in adherence to and internalizationof S. uberis into bovine mammary epithelial cells, indicating theimportance of SUAM as a significant S. uberis virulence factor.

[0027] (8) A theoretical DNA sequence of SUAM was determined andconfirmed by PCR and restriction digests.

[0028] (9) The “true” DNA sequence encoding for SUAM was elucidated andfound to have 99% homology to the theoretically elucidated SUAM DNA.

[0029] It is conceived that this single virulence factor (SUAM) plays acritical role in the pathogenesis of streptococcal mastitis byfacilitating bacterial adherence to bovine mammary epithelial cells. Itis conceived that S. uberis expresses SUAM and uses LF in milk and/or onthe epithelial cell surface to adhere to mammary epithelial cells. It isfurther conceived that antibodies that bind to SUAM or pepSUAM may beused to diagnose infections due to S. uberis or other streptococci or totreat infections due to S. uberis or other streptococci. It is furtherconceived that nucleic acid sequences that encode SUAM or pepSUAM may beused diagnostically or in the production of anti-streptococcal vaccines.It is further conceived that the SUAM and pepSUAM polypeptides of theinvention may be used to in the production of antisera or vaccines tocombat diseases due to S. uberis or other streptococci.

[0030] In one embodiment, the invention is a polypeptide comprising anamino acid sequence of at least 6 sequential amino acids of pepSUAM(MTTADQSPKLQGEEA), designated herein as Seq. ID No. 4, wherein anantibody that binds to the polypeptide inhibits adherence to and/orinternalization of S. uberis into bovine mammary epithelial cells. Forexample the 6 sequential amino acids of the polypeptide of the inventionmay be amino acids 1 to 6, 2 to 7, 3 to 8, 4 to 9, 5 to 10, 6 to 11, 7to 12, 8 to 13, 9 to 14, or 10 to 15 of Seq. ID No. 4 pepSUAM.

[0031] Preferably, the polypeptide of this embodiment of the inventioncomprises an amino acid sequence of more than 6 sequential amino acidsof pepSUAM of Seq. ID No. 4, for example, 7, 8, 9, 10, 11, 12, 13, 14sequential amino acids, or the entire 15 amino acid sequence of Seq. IDNo. 4. The polypeptide of the invention may further contain additionalamino acids to the amino terminal or carboxy terminal sides of thesequence that is a portion or all of pepSUAM. For example, thepolypeptide of the invention may contain at its amino terminal end theamino acids DD, which are present at the amino terminal end offull-length SUAM.

[0032] The polypeptide may be used to elicit antibodies which may beused to diagnose infections due to SUAM-expressing organisms such asStreptococcus, like S. uberis. The polypeptide may also be used toelicit an immune response in an animal or human that is susceptible toinfection by an organism that contains a surface antigen that will bindto an antibody that binds to the polypeptide of the invention. Thus, thepolypeptide of the invention may be useful as a vaccine againstinfection due to Streptococcus, such as S. uberis, S. pyogenes, S.agalactiae, or S. dysgalactiae.

[0033] In another embodiment, the invention is an isolated SUAM proteinpreferably having the amino acid sequence shown in FIG. 4 or FIG. 6 anddesignated herein as Seq. ID No. 2 or Seq. ID No. 15, respectively.

[0034] In another embodiment, the invention is a polypeptide derivedfrom SUAM protein, which may be isolated by the method described belowand which comprises the sequence of amino acids MTTADQSPKLQGEEA, Seq. IDNo. 4.

[0035] The invention also includes polypeptides that are substantiallyhomologous with the pepSUAM polypeptide or SUAM protein and polypeptidesderived therefrom, as described above. As used in this context, the term“substantially homologous” means that the amino acid sequence shares atleast 50%, such as at least 60%, preferably at least 70%, morepreferably at least 80%, and most preferably at least 90% amino acididentity with the pepSUAM or SUAM protein or polypeptides derivedtherefrom and wherein an antibody that binds to the polypeptide inhibitsthe adherence and/or the internalization of S. uberis to bovine mammaryepithelial cells.

[0036] In another embodiment, the invention is an antibody thatselectively binds to an amino acid sequence of any 6 to 15 sequentialamino acids of pepSUAM, as described above. Preferably, the antibodyinhibits the adherence and/or the internalization of S. uberis to bovinemammary epithelial cells. The antibody may be a monoclonal or polyclonalantibody and may be used diagnostically or therapeutically.

[0037] In another embodiment, the invention is an antibody thatselectively binds to the SUAM polypeptides or proteins of the invention.Preferably, the antibody inhibits the adherence and/or theinternalization of S. uberis to bovine mammary epithelial cells. Theantibody may be a monoclonal or polyclonal antibody and may be useddiagnostically or therapeutically.

[0038] In another embodiment, the invention is an isolated nucleic acidsequence that encodes the pepSUAM polypeptide. Preferably, the nucleicacid sequence comprises the sequence shown in underline and in bold inFIG. 3, and designated Seq. ID No. 5:ATGACAACTGCTGATCAATCACCTAAATTACAAGGTGAAGAAGCA.

[0039] In another embodiment, the invention is an isolated nucleic acidsequence that encodes the SUAM protein. Preferably, the nucleic acidsequence comprises either of the sequence shown in FIGS. 3 or 5,designated Seq. ID No. 1 and Seq. ID No. 3, respectively. Morepreferably, the nucleic acid sequence comprises the sequence fromnucleotide 317 to nucleotide 2836 of Seq. ID No. 1 or from nucleotide289 to nucleotide 2808 of Seq. ID No. 3. Most preferably, the nucleicacid sequence comprises the sequence from nucleotide 311 to nucleotide2836 of Seq. ID No. 1 or nucleotide 283 to nucleotide 2808 of Seq. IDNo. 3.

[0040] Also included in the isolated nucleic acid sequences of theinvention is a nucleic acid sequence that will hybridize under highlystringent conditions, for example at 3×SSC at 65° C. and preferably at6×SSC at 65° C., to the complement of the above specifically describednucleic acid sequences.

[0041] In another embodiment, the invention is a method for immunizingan animal or human with an antigen against a bacterial organism. Inaccordance with the method of the invention, the polypeptide of theinvention or the SUAM polypeptide is administered to an animal or humansubject by any suitable means such as by injection or intramammaryinfusion and the subject is thereby caused to produce antibodies thatselectively bind thereto, which antibodies inhibit bacteria that bind tolactoferrin from adhering and/or internalizing to cells and/or enhanceclearance of bacterial pathogens. In this way, the ability of themicroorganism to cause disease is reduced.

[0042] In another embodiment, the invention is a primer selected fromthe group of (a) 5′-GTC ATT TGG TAG GAG TGG CTG-3′, (Seq. ID No 6) (b)5′-TGG TTG ATA TAG CAC TTG GTG AC- (Seq. ID No 7) 3′, (c) 5′-GGA TGA CATGAC AAC TGC TGA TC- (Seq. ID No 8) 3′, (d) 5′-CAA TTG TCA GCA CGT CTCTGT AC- (Seq. ID No 9) 3′, (e) 5′-CTT GGA ACT GGT GTT GGT ATG G- (Seq.ID No 10) 3′,, and (f) 5′-CAG GTG TTA CTT CAG GTG CTA C- (Seq. ID No 11)3′.

[0043] Preferably, the primers are grouped in pairs with primers (a) and(b) being paired as a forward and reverse PCR primer, respectively,primers (c) and (d) being paired as a forward and reverse PCR primer,respectively, and primers (e) and (f) being paired as a forward andreverse PCR primer, respectively.

[0044] The primers and primer pairs of the invention are useful, forexample, in identifying microorganisms that produce SUAM or apolypeptide molecule having a high degree of homology to SUAM, such as70% or more homology. As such, the primers of the invention may be usedto diagnose the presence of an infection with a SUAM polypeptide, orSUAM-like polypeptide, producing microorganism. It is conceived that ananimal or human patient that is diagnosed in this manner may be treatedwith administration of the polypeptide of the invention to induce animmune response against such microorganism.

[0045] Following is a list of possible applications of variousembodiments of the invention. This list is not intended to be allinclusive as those skilled in the art will understand that additionaluses exist for the invention.

[0046] I. Antibodies to SUAM and pepSUAM

[0047] A. Commercial use

[0048] Diagnostic

[0049] Microbiology: immuno-fluorescence, card-test for preliminaryconfirmation (including cow-side rapid tests using milk from cows withmastitis)

[0050] Serology: Agglutination/precipitation tests (cow-side rapidtests), ELISA

[0051] Diagnostic enrichment of bacteria from crude samplesTreatment/Prevention

[0052] Therapy for cows with mastitis (systemic/intramammary) Preventionfor new cows introduced to a farm with history of S. uberis infection

[0053] Intramammary preparations for cows near parturition

[0054] B. Research use

[0055] 1. Isolation/purification of SUAM

[0056] 2. In vitro pathogenicity assays

[0057] 3. Recombinant protein expression (monitoring and isolation)

[0058] 4. Mutant detection

[0059] 5. Immuno-histochemistry

[0060] 6. Western blot

[0061] 7. Immunoprecipitation for protein/protein interaction studies

[0062] 8. Steric inhibition studies

[0063] II. Suam Protein

[0064] A. Commercial use

[0065] 1. Vaccine production

[0066] 2. Antisera production

[0067] 3. Protein as antigen component of multivalent vaccine

[0068] B. Research use

[0069] 1. Antisera production

[0070] 2. Experimental vaccination studies

[0071] 3. Protein as antigen component of multivalent vaccine

[0072] 4. Protein as ligand in affinity purification of bovinelactoferrin

[0073] III. pepSUAM

[0074] A. Commercial use

[0075] 1. Vaccine production

[0076] 2. Antisera production

[0077] 3. Peptide as antigen component of multivalent vaccine

[0078] 4. Peptide as competitive inhibitor of adhesion/invasion inintramammary preps

[0079] B. Research use

[0080] 1. Antisera production

[0081] 2. Experimental vaccination studies

[0082] 3. Peptide as component of multivalent vaccine

[0083] 4. Peptide as ligand in affinity purification of BovineLactoferrin

[0084] IV. Suad DNA Sequence

[0085] A. Commercial use

[0086] Diagnostic

[0087] 1. Probes

[0088] 2. PCR (alternative primers design)

[0089] 3. Cow-side rapid test (i.e., cantilever)

[0090] Prevention of mastitis

[0091] 1. Recombinant expression for vaccine production (baculo-viruscloning and expression)

[0092] 2. DNA vaccines (cloning into retro-virus vectors orAgrobacterium tumefaciens)

[0093] 3. Cloning and expression in vitro for vaccine production

[0094] B. Research use

[0095] 1. Probes

[0096] 2. PCR (alternative primers design)

[0097] 3. Real time PCR for selection and identification of strains

[0098] 4. DNA microarrays/differential display (to identify and studyfactors that enhance or repress SUAM expression)

[0099] 5. Site directed mutagenesis

[0100] 6. Production of avirulent carrier strain for this or any otherexpressed protein vaccine.

[0101] V. Suam PCR Primers

[0102] A. Commercial use

[0103] Diagnostic

[0104] 1. PCR amplification products detected by any means

[0105] 2. Real time PCR (taq-man, beacons, etc.)

[0106] 3. Probes

[0107] B. Research use

[0108] 1. PCR Detection

[0109] 2. Real time PCR (taq-man, beacons, etc.)

[0110] 3. Probes for southerns, reverse transcriptase protection assays,etc.

[0111] 4. Cloning and expression

[0112] The invention is further illustrated in the followingnon-limiting examples.

EXAMPLE 1

[0113] Identification of Streptococcus uberis Lactoferrin-bindingProteins (prior art), (described in Fang, W., and S. P. Oliver, FEMSMicrobiol. Lett. 176:91) (1999)

[0114] Experiments were conducted to examine binding of lactoferrin (LF)by strains of S. uberis causing bovine mastitis and to identify proteinsfrom the bacteria involved in LF-binding. Four strains of S. uberisisolated originally from dairy cows with mastitis and S. uberisATCC13387 (American Type Culture Collection, Rockville, Md.) wereevaluated. After growth, bacterial cultures were washed and split intotwo equal portions: one for incubation in milk and the other inphosphate buffered saline (PBS) (as controls). Bacterial surfaceproteins from pellets were extracted using 0.2% sodium dodecyl sulfate(SDS) and electrophoresed. Gels were silver-stained or transferred ontonitrocellulose membranes for immunoblotting using rabbit anti-bovine LFantibody and HRP (horseradish peroxidase)-conjugated donkey anti-rabbitIgG antibody as probes.

[0115] Biotin-avidin-based binding assay (BABA) and ELISA-based bindingassay were carried out on immobilized S. uberis microplates. LF frombovine milk and transferrin (TF) from bovine plasma were biotinylated.For the BABA assay, serial 2-fold dilutions of biotinylated LF wereadded into microplate wells, incubated, washed, and probed withHRP-NeutrAvidin. The ELISA-based assay was essentially the same as BABAexcept that serial 2-fold dilutions of unlabelled LF were substitutedfor biotinylated LF. Rabbit anti-bovine LF antibody and HRP-conjugateddonkey anti-rabbit IgG antibody were used as probes. Inhibition ofLF-binding by unlabelled LF, TF, mannose, galactose, and lactose werealso tested using BABA and ELISA.

[0116] Polypeptides that bound to LF were identified by SDS-PAGE andwestern blot analysis of bacterial surface proteins. Blots were probedsequentially with LF, rabbit anti-bovine LF antibody, and HRP-conjugateddonkey anti-rabbit IgG antibody. At least two proteins in each strain ofS. uberis were identified as lactoferrin-binding proteins. Theseincluded 52 and ˜112 kDa bands in 4 of 5 strains evaluated. One strainof S. uberis did not have the 112 kDa protein band, however, this strainproduced a higher molecular weight protein (134 kDa) which also bound toLF.

[0117] The microplate-based assay systems demonstrated that S. uberisbound to purified LF. These studies provided evidence that S. uberisbinds to LF in milk and that at least two proteins from S. uberissurface molecules are involved in LF-binding.

EAXMPLE 2

[0118] Effect of Lactoferrin on Adherence of Streptococcus uberis toBovine Mammary Epithelial Cells (prior art) (described in Fang, W., R.A. Almeida, and S. P. Oliver, Am. J. Vet. Res. 61:275) (2000)

[0119] A series of experiments were conducted to determine effects of LFon adherence of S. uberis to mammary epithelial cells. Three strains ofS. uberis were used. In the first experiment, we investigated the effectof LF on adherence of S. uberis to bovine mammary epithelial cells.Sterile LF in Dulbecco's Modified Eagle Medium (DMEM) or milk (0.5 ml)and 0.5 ml of bacterial suspension containing 1-2×10⁸ cfu/ml in DMEMwere added to bovine mammary epithelial cell line (MAC-T) monolayers.Final concentrations for LF were 0, 0.01, 0.1 and 1 mg/ml. Those formilk were 0, 12.5%, 25% and 50%. Bacteria were allowed to adhere toMAC-T cells and supernatants were then aspirated and diluted forbacterial counting. Monolayers were washed and lysed, and cell lysateswere 10-fold diluted for bacterial counting. Streptococcus uberiscultures were also pretreated with LF (1 mg/ml) or milk (100%) for 1 h.Bacterial suspensions in PBS (without LF or milk) were included ascontrols. Bacteria were then washed and adjusted to 1-2×10⁸ cfu/ml foradherence assays.

[0120] To test the effect of anti-bovine LF antibody on adherence, a S.uberis strain was pretreated with LF or milk as described above andexamined for its adherence to MAC-T cells in presence of differentdilutions of rabbit anti-bovine LF antibody. For the microscopicadherence assay, strains of S. uberis were labeled with fluoresceinisothiocyanate (FITC). FITC-labeled bacteria were resuspended in DMEM.Sterile LF in DMEM (0.15 ml) and FITC labeled bacteria (0.15 ml) wereadded to wells of chamber slides containing confluent MAC-T cellmonolayers. After incubation, bacterial supernatants were removed, andslides were washed and examined microscopically.

[0121] All strains of S. uberis evaluated bound to LF in milk and topurified LF. LF and milk enhanced adherence of S. uberis to MAC-T cellswhen present in the test medium (P<0.05-0.01) except for one strain ofS. uberis. Pretreatment of bacteria with LF and milk increased adherenceof one strain of S. uberis (P<0.01), but not the other two strains. Itis conceived that differences between LF or milk pretreatment andpresence of LF or milk in the medium could partially account for thedifferent results. Because LF is synthesized and secreted by mammaryepithelial cells and also binds to mammary epithelial cells, it isconceived that the presence of LF in the test medium might enhance thepotential of LF as a bridging molecule between bacteria and MAC-T cells,thus increasing adherence. Additionally, differences of intrinsicsurface properties among S. uberis strains might affect theirinteraction with LF as well as with MAC-T cells. There were differencesamong these S. uberis strains in hydrophobicity. Two strains of S.uberis were more attracted to hexadecane as well as to MAC-T cells thanwas a third strain of S. uberis.

[0122] The involvement of milk in the adherence of S. uberis to MAC-Tcells may be more complicated than that of purified LF because of thecoexistence of other milk components that may also play a part inbacterial interactions with epithelial cells. For example, ourlaboratory demonstrated and reported that adherence to extracellularmatrix proteins, particularly collagen, enhanced adherence andinternalization of S. uberis to bovine mammary epithelial cells and thatpresence of these host proteins up-regulated expression of ligands forcollagen. Therefore, LF is not the only host protein that binds to S.uberis. However, our data indicate specific involvement of LF inadherence since addition of rabbit anti-bovine LF antibody significantlydecreased adherence of LF or milk-pretreated bacteria to MAC-T cells(P<0.01) at dilutions below 1:500 for LF and 1:100 for milk.

[0123] The results of these studies indicate that LF functions as abridging molecule between S. uberis and bovine mammary epithelial cellsand facilitates adherence of this mastitis pathogen to the host cells.

EXAMPLE 3

[0124] Investigation of Influence of Strain of S. uberis on theEnhancing Effect of LF on Adherence and Internalization to MammaryEpithelial Cells

[0125] To further investigate a possible strain influence on theenhancing effect of LF on adherence and internalization to mammaryepithelial cells, additional studies were conducted. In these studies,six strains of S. uberis isolated originally from milk of dairy cowswith mastitis were used. Bacteria were pretreated with LF (ICN, Aurora,Ohio), 21.4% iron saturation and 97.5% protein content, 1 mg/ml) for 1 hat 37° C., washed 3 times with PBS (pH 7.4), resuspended in DMEM andcocultured with MAC-T cells for 1 h. After incubation, supernatants wereremoved, monolayers were washed and either lysed with trypsin/tritonsolution to determine total cell associated bacteria or treated withantibiotic solution to determine internalization of bacteria intomammary epithelial cells. For the latter, after 2 h of incubation,antibiotic solution was removed, monolayers were washed 3 times with PBSand cells were lysed with trypsin/triton solution. Colony forming unitsper ml (CFU/ml) in lysates were determined using standard colonycounting techniques. Although differences in adherence andinternalization were detected among strains, addition of LF causedsignificantly greater adherence or internalization to mammary epithelialcells of all strains of S. uberis evaluated.

[0126] It is conceived that adherence and internalization are not twoseparate independent events. Adherent bacteria are quickly internalizedthrough an endocytic-like mechanism, where receptors for the “bridging”proteins are recycled and exposed in or on the host cell surface. Thekinetics of these events has been described as a chain reaction whereadherence promotes internalization. Therefore, higher concentrations ofthe “bridging” protein results in increased adherence that in turn leadsto increased internalization rather that reversal of adherence. Thus,increased binding of LF by S. uberis mediated by a lactoferrin bindingprotein (SUAM) results in increased bacterial internalization intomammary epithelial cells.

[0127] Thus, it is conceived that, by a mechanism referred to herein as“molecular bridging” LF possesses different binding domains, a bindingdomain for the host cell and another binding domain for S. uberis (seethe schematic presented in FIG. 2 “Diagram 1”). The interaction betweenhost cell receptor and the host-domain region in S. uberis bound LFallows contact of the bacterium and host cell surface membrane resultingin adherence. The interaction between LF and its host cell receptortriggers arrangements on the host membrane that initiate theinternalization of the bacterium into the host cell.

EXAMPLE 4

[0128] Isolation, Purification and N-terminal Amino-acid Sequencing ofStreptococcus uberis Adhesion Molecule (SUAM)

[0129] A study was conducted to compare potential differences in theefficiency of extraction of SUAM with mutanolysin or SDS by SDS-PAGE andWestern blotting. Four strains of S. uberis were used. Bacterial surfaceproteins from cell pellets were extracted from 0.2% SDS in PBS (pH 7.2)following the method described by Fang and Oliver (1999). Each strain ofS. uberis was grown in THB (Todd-Hewitt Broth) (Difco Laboratories,Detroit, Mich.) at 370C overnight. After centrifugation, bacteria wereresuspended in PBS. Bacterial pellets were washed three times withsterile PBS, and surface proteins were extracted using 0.2% SDS (sodiumdodecyl sulfate) (Bio-Rad Laboratories, Hercules, Calif.; 30 mg wetweight of bacteria per 100 μl of 0.2% SDS) for 1 h at 37° C.

[0130] In the mutanolysin extraction method, a modified procedure wasused. Bacterial cells were suspended (1 g/2 ml) in 50 mM phosphatebuffer (pH 7.2), containing 0.5 M sucrose and 10 mg/ml lysozyme (Sigma,St. Louis, Mo.). The resulting suspension was divided into 2 ml aliquotsand 250 units of mutanolysin (N-acetylmuramidase, Sigma) were added peraliquot. The suspension was shaker incubated for 1 h at 37° C. Bacteriawere pelleted by centrifugation and supernatants of each were removedand stored at −20° C.

[0131] Extracted bacterial surface proteins (10 μg/lane) wereelectrophoresed on 10% SDS-PAGE. Gels were stained with Coomassiebrilliant blue or transferred onto nitrocellulose membrane usingTrans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). Unboundsites on blots were blocked with 3% casitone. Blots were probed with LF(ICN, 5 μg/ml) in PBS Tween 20 (PBST) containing 0.1% casitone for 6 hat 4° C., followed by four washes with PBST. Procedures for furtherprobing of blots with rabbit anti-bovine LF antibody and HRP-conjugateddonkey anti-rabbit IgG antibody were as described previously (Fang andOliver, 1999). Blots without probing with LF and rabbit anti-bovine LFantibody were included as negative controls.

[0132] When surface proteins were extracted with 0.2% SDS detergent andevaluated by SDS-PAGE, 110 kDa and 112 kDa protein bands were extractedmore efficiently compared to the mutanolysin extraction method. InWestern blot analysis, the intensity of SUAM bands in SDS extracts,particularly 110 and 112 kDa, were much stronger than those ofmutanolysin extracts. Results of this study indicate that SDS extractsproteins of interest (110 kDa and 112 kDa) more efficiently and is apreferred method for SUAM purification and subsequent characterization.

EXAMPLE 5

[0133] Iron Availability Influences Expression of SUAM

[0134] A study was conducted in which the effect of an iron chelator onexpression of S. uberis was evaluated. Strains of S. uberis were growneither in THB or THB treated with the iron chelator 2,2-dipyridyl andsurface proteins from bacterial pellets were analyzed by Western blotusing LF as a probe and rabbit anti-bovine LF antibody. Western blotanalysis showed two major bands of 110 KDa and 112 KDa, respectively,with LF-binding activity. In addition, LF-binding activity decreased inthe presence of an iron chelator which indicates that iron in the mediuminfluences expression of SUAM.

EXAMPLE 6

[0135] Purification of SUAM

[0136] Thirty ml of PBS (pH 7.4) containing 30 mg of SDS-extracted S.uberis surface proteins were loaded into a bovine LF-coupledCNBr-activated Sepharose 4B column. SDS-extracted surface proteins wereincubated with shaking for 2 h at 4° C. with 7 ml of Sepharose 4Bcovalently linked to bovine LF (ICN, 21.4% iron saturation and 97.5%protein content). The LF-Sepharose 4B slurry was loaded into achromatography column (1.25 cm×9 cm; total volume 70 ml) (Pfizer, NewYork, N.Y.). The column was subsequently washed with 10 volumes of TBS(50 mM Tris-HCl (pH 7.4) +150 mM NaCl containing 0.1% Triton-X 100) toremove nonspecific-binding proteins using a peristaltic pump at a flowrate of 1 ml/min until absorbance at 280 nm approached zero. The columnwas eluted with a sodium chloride gradient from 0.1 M to 1 M NaCl inTBS. Fractions (10 ml/fraction) were analyzed by absorbance at 280 nm,SDS-PAGE and Western blot using rabbit anti-bovine LF antibodies andbiotinylated LF as probes. Fractions containing SUAM were pooled,dialyzed against PBS and stored at −70° C. until use.

[0137] Analysis of fractions revealed the presence of a protein infraction number 14 to 32 eluted at 0.5M NaCl. The molecular mass wasestimated to be ˜112 kDa using Gel Scan (Corbett Research, Mortlake,NSW, Australia). Results from SDS-PAGE and Western blot analysisindicated that this band had LF-binding affinity. The yield of purifiedSUAM was 20 μg/ml (total 10 ml) from 3 liters of THB-grown S. uberis.

EXAMPLE 7

[0138] N-terminal Amino Acid Sequence of the 112 kDa SUAM

[0139] Excised PVDF membrane (PerkinElmer Life and Analytical Sciences,Inc., Boston, Mass.) containing ˜112 kDa SUAM band was analyzed. Theprotein was sequenced on an Applied Biosystems model 477A sequencer(Applied Biosystems, Foster City, Calif.) equipped with on-line PTHanalysis using the regular program O3RPTH. The PTH-derivatives wereseparated by reverse-phase HPLC over a Brownlee C-18 column (220×2.1mm). The initial yield for the coupling step was calculated from theamount of PTH-derivatives present in the first cycle and by the amountof protein spotted. As a standard marker for amino acid sequence, therepetitive yield from myoglobin was determined from peak heights ofvaline, leucine, and glutamic acid according to the positions. Therepetitive yield from β-lactoglobulin was calculated for leucine,isoleucine, and valine residues. The N-terminal amino acid sequence ofSUAM was D D M T T A D Q S P K L Q G E E A (T/A) L (I/A) (V/K) (Seq. IDNo. 12).

[0140] The above procedures were repeated and an identical amino acidsequence was obtained. A protein-nucleic acid TBLASTN search (NCBI) andSwissprot amino acid data bank search were used to align theSUAM-terminal amino acid sequence with previously sequenced genes andproteins. No similarities were found establishing that the bacterialSUAM protein is a novel protein.

EXAMPLE 8

[0141] Identification of Lactoferrin Binding Proteins in Streptococcusdysgalactiae subsp. dysgalactiae and Streptococcus agalactiae Isolatedfrom Cows with Mastitis (prior art) (described in Park, H. M., R. A.Almeida, and S. P. Oliver, FEMS Microbiol. Lett. 207:87 (2000))

[0142] This paper demonstrates the presence of lactoferrin-bindingproteins in two major bovine mammary pathogens, Streptococcusdysgalactiae subsp. dysgalactiae (S. dysgalactiae) and Streptococcusagalactiae.

[0143] Three strains of S. dysgalactiae and five strains of S.agalactiae were used to identify lactoferrin-binding proteins (LBPs).LBPs from extracted surface proteins were detected by polyacrylamide gelelectrophoresis and Western blotting. All strains of S. dysgalactiaeevaluated had 52 kDa and 74 kDa protein bands. All strains of S.agalactiae evaluated had 52 kDa, 70 kDa and 110 kDa protein bands. Inaddition, a 45 kDa band was detected in two of five S. agalactiaestrains evaluated. This study demonstrated that S. dysgalactiae and S.agalactiae of bovine origin contain at least two major LBP's. Thus,LBP's are present in several Streptococcus species that cause mastitisin dairy cows.

EXAMPLE 9

[0144] Binding of Bovine Lactoferrin to Streptococcus dysgalactiaeSubsp. dysgalactiae Isolated form Cows with Mastitis (prior art) (Park,H. M., R. A. Almeida, D. A. Luther, and S. P. Oliver, FEMS Microbiol.Lett. 208:35 (2000))

[0145] Three strains of S. dysgalactiae subsp. dysgalactiae (one ofwhich is strain ATCC 27957) were used to determine if bovine lactoferrin(LF) binds to bacterial cells by biotin avidin binding assay (BABA),enzyme-linked immunosorbent assay (ELISA), and binding inhibition assay.Binding assays revealed that all strains of S. dysgalactiae subsp.dysgalactiae (S. dysgalactiae) evaluated in this study bound to LF,although some differences in LF binding capability among strains andbetween methods used were detected. Binding of LF was not inhibited bytransferrin (TF) and LF moiety molecules (mannose, galactose, andlactose) but by LF. This study demonstrates that S. dysgalactiae bindsto bovine LF in a specific manner.

EXAMPLE 10

[0146] Production of Antibodies against SUAM (whole protein) and to aSynthetic Peptide (pepSUAM) Encompassing 15 Amino Acids Near theN-terminus of SUAM.

[0147] SUAM antibodies were needed to test the biological role of SUAMon adherence to and internalization of S. uberis into bovine mammaryepithelial cells, and to test protective effects of SUAM antibody onthese in vitro approaches. To obtain antibodies, purified SUAM asdescribed in Example 6 was sent to Quality Bioresources Inc. (QBI,Seguin, Tex.) for custom antibody production. For production ofantibodies against SUAM, ˜300 μg of purified protein was used toimmunize two rabbits. For production of antibodies against SUAM-derivedpeptide (pepSUAM), Bethyl Laboratories, Inc. (Montgomery, Tex.)synthesized the selected peptide based on the N-terminal amino acidsequence M T T A D Q S P K L Q G E E A (Seq. ID No. 4). All peptideswere HPLC purified and conjugated to KLH for immunization. PepSUAMinduced a high immune response with production of immunologic responsewhich yielded 20 mg of affinity purified antibody.

EXAMPLE 11

[0148] Cross-reactivity of pepSUAM and SUAM Antibodies with SeveralStrains of S. uberis.

[0149] To ensure that SUAM is not a rare protein found only in onestrain of S. uberis, and that research or prophylactic productsdeveloped will have broad significance, several strains of S. uberisfrom diverse locations were tested by Western blotting. Strainsevaluated were from Tennessee, Colorado, Washington and New Zealand. Thedifferent S. uberis strains were cultured overnight in Todd Hewitt brothand surface proteins were extracted in Laemmli sample buffer. SDS-PAGEpolyacrylamide gels (7.5%) were electrophoresed followed by transfer tonitrocellulose membranes. They were blocked in PBSTG (phosphate bufferedsaline, 0.05% (v/v)Tween-20, and 0.1% (w/v) porcine gelatin) for 1 h.Affinity purified rabbit anti-pepSUAM and rabbit anti-SUAM antibodieswere diluted in PBSTG (1:2000) and blots treated for 1.5 h. Followingwashing of blots with several changes of PBST, a 1:2000 dilution inPBSTG of peroxidase-conjugated affipure F(ab′)2 fragment donkeyanti-rabbit IgG (H+L) was applied. The SUAM protein band was revealedwith the peroxidase substrate 4CN (4-chloro-1-naphthol). The presence ofa single dominant band on a blot of total S. uberis detergent extractedsurface proteins attests to the specificity of the antibodies. The 112kDa SUAM protein band is clearly visible. These results establish thatSUAM is a ubiquitous protein in S. uberis strains and that pepSUAM mayplay a role as a universal immunogen to protect against S. uberismastitis.

EXAMPLE 12

[0150] Cross-reactivity of pepSUAM and SUAM Antibodies with S.agalactiae, S. dysgalactiae Subsp. dysgalactiae, and Streptococcuspyogenes.

[0151] Cross-reactivity of rabbit anti-SUAM whole protein antibodies andrabbit anti-pepSUAM antibodies between different Streptococcus specieswas investigated. Strains of S. dysgalactiae subsp. dysgalactiae, S.agalactiae (from animals and humans), and Streptococcus pyogenes werecultured overnight in Todd Hewitt broth and bacterial surface proteinswere extracted in Laemmli sample buffer. SDS-PAGE polyacrylamide gels(7.5%) were electrophoresed followed by transfer to nitrocellulosemembranes. They were blocked in PBSTG (phosphate buffered saline, 0.05%(v/v)Tween-20, and 0.1% (w/v) porcine gelatin) for 1 h. Affinitypurified rabbit anti-pepSUAM and rabbit anti-SUAM antibodies werediluted in PBSTG (1:2000) and blots treated for 1.5 h. The nexttreatment after washing blots with several changes of PBST was a 1:2000dilution in PBSTG of peroxidase-conjugated affipure F (ab′) 2 fragmentdonkey anti-rabbit IgG (H+L). The SUAM protein band was revealed withthe peroxidase substrate 4CN (4-chloro-1-naphthol). Western blot resultsshowed cross reaction of pepSUAM and SUAM antibodies with proteins ofother Streptococcus species, including the human pathogen S. pyogenes.The cross reaction with other proteins or protein fragments indicatesthat SUAM and its functions are conserved or partially conserved betweenStreptococcus species and that a vaccine based upon SUAM would havebroad application.

EXAMPLE 13

[0152] Inhibitory Effect of SUAM and pepSUAM Antibodies on Adherence andInternalization of S. uberis to Bovine Mammary Epithelial Cells.

[0153] Two strains of S. uberis isolated from cows with clinicalmastitis were incubated with increasing concentrations of SUAM andpepSUAM antibodies, co-cultured with bovine mammary epithelial cells andadherence of S. uberis to and internalization of S. uberis into mammaryepithelial cells measured.

[0154] A bovine mammary epithelial cell line (MAC-T) was used. MAC-Tcells were cultured in cell growth medium (CGM) in 24-well plates andincubated in 5% CO₂/balance air at 37° C. Monolayers were checked dailyfor confluence.

[0155] Two S. uberis strains isolated from cows with mastitis were used.For adherence and internalization assays, bacteria stored at −70° C.were thawed in a 37° C. water bath, streaked onto blood agar plates, andincubated for 16 h at 37° C. Bacteria were then inoculated intoTodd-Hewitt broth (THB, Difco, Detroit, Mich.) for 2 h at 37° C.Bacterial suspensions were diluted in CGM to a concentration of 10⁷bacteria per ml.

[0156] Each of the two strains of S. uberis was preincubated withseveral dilutions of SUAM and pepSUAM antibodies for 1 h at 37° C. Afterincubation, bacterial suspensions were washed three times to removeunbound antibodies and co-cultured with MAC-T cells for 2 h at 37° C. in5% CO2: 95% air (vol/vol). In order to enumerate bacteria associatedwith MAC-T cells (adherent+internalized bacteria), MAC-T cells werewashed 3 times to remove unbound bacteria and lysed with trypsin andtriton. MAC-T cell lysates were 10-fold serially diluted, seeded intriplicate on blood agar plates, and incubated overnight at 37° C. Afterincubation, individual colonies were counted and expressed as colonyforming units per ml (CFU/ml) of S. uberis.

[0157] In order to discriminate between S. uberis that adhered to theMAC-T cell surface from those that were internalized into MAC-T cells,an internalization assay was performed in parallel wells and under thesame culture conditions as described for the adherence assay. Theinternalization assay was similar to the adherence assay with theexception that an antibiotic treatment directed to destroy bacteria thatwere not internalized was performed before lysing MAC-T cells. Followingthis, MAC-T cells were washed extensively, lysed as described before,and bacteria that were internalized were enumerated as described for theadherence assay. The number of adherent bacteria was calculated bysubtracting the number of internalized bacteria from MAC-Tcell-associated bacteria.

[0158] Pretreatment with SUAM (FIG. 1A&B) or pepSUAM (FIG. 1C&D)antibodies reduced adherence and internalization of S. uberis to mammaryepithelial cells. The greatest adherence and internalization of S.uberis was observed when S. uberis was not pretreated with SUAM orpepSUAM antibodies. The lowest adherence and internalization of S.uberis was detected when higher concentrations of antibodies were used.FIG. 1A-D show a dilution effect on adherence and internalization, whichconfirms the inhibitory effect of SUAM and pepSUAM antibodies onadherence to and internalization of S. uberis into MAC-T cells.

[0159] Results from this experiment showed the inhibitory effect of SUAMand pepSUAM antibodies on adherence and internalization of S. uberisinto MAC-T cells and indicate the value of SUAM and pepSUAM asimmunogens for controlling this economically important disease of dairycows.

EXAMPLE 14

[0160] Theoretical Elucidation of SUAM DNA Sequence and Confirmation byPCR and Restriction Digest.

[0161] Theoretical elucidation of the DNA sequence from the pepSUAMamino acid sequence permitted DNA synthesis of the SUAM gene using PCRtechniques. The pepSUAM amino acid sequence (MTTADQSPKLQGEEA) (Seq. IDNo. 4) was used to search a S. uberis genomic database (Wellcome TrustSanger Institute) to identify a single fragment of the genome, alsoknown as “contig”, that matched the DNA sequence of pepSUAM amino acids.The match for pepSUAM was 100% for this DNA contig and this was the onlymatch of this quality in the entire existing S. uberis genomic database.From this DNA contig, several PCR primers were designed and used in PCRreactions to obtain a unique DNA fragment. Subsequent analysis of thisPCR fragment showed physical and DNA sequence characteristics similar tothat of the elucidated SUAM gene. These results indicate that a uniqueand single gene of the S. uberis genomic sequence is responsible forcoding SUAM and that we generated unique PCR primers and defined PCRconditions for the synthesis of SUAM.

[0162] Using the ExPASy Home Page Translate Tool (Swiss Institute ofBioinformatics), the S. uberis genomic contig DNA sequence wastranslated to amino acid sequences, in all possible reading frames. Onlyone of the six possible translations contained an open reading frame (anarea without stop codons) long enough to code for the S. uberis protein.This sequence was checked using a BLAST search against the entireNational Center for Biotechnology Information (NCBI) genomic databaseand appears to be unique, with only partial segments showing homology.

[0163] The sequence shown in FIG. 3 is the hypothetical SUAM genesequence with some additional sequence included before and after, 3,041nucleotides. This sequence is designated as Seq. ID No. 1. The codingregion for the N-terminal sequence begins at nucleotide 311 and ends at376 (underlined). The coding region for the peptide used to generateantibody is from nucleotide 317 to 360 (bold). The open reading frame,i.e. gene, ends at the stop/termination codon represented by TAA,nucleotides 2837 to 2839.

[0164]FIG. 4 shows the translation of the nucleotide sequence of Seq. IDNo. 1 in the correct reading frame. This amino acid sequence isdesignated Seq. ID No. 2. The N-terminal sequence segment is underlinedand the peptide used to generate the antibody to pepSUAM is underlinedand bold. The end that corresponds to the above sequence (bold TAA inFIG. 3) is marked by the dash following the bold GKK, which would becoded for by GGCAAAAAA.

[0165] This selected coding region was used to design primers for itsamplification by PCR. Three separate pairs of primers that bound to sixindividual sites were designed to generate three slightly differentfragments from this same gene. These primers successfully generated PCRproducts of the predicted length. This provides very strong evidencethat this gene is present in the strain of S. uberis (S. uberis UT888)from which the S. uberis protein (SUAM) was purified and the N-terminalpeptide sequence was determined. One of these primers was homologous tothe coding region for the N-terminal sequence providing further supportthat the correct gene was amplified.

[0166] In an effort to determine additional amino acid/protein andnucleic acid/DNA sequence, three independent pairs of PCR primers weredesigned from the S. uberis genomic database sequence, contig sub114a06.TABLE 1 Name, nucleotide composition and expected PCR product size. SEQID PRODUCT NAME PRIMER NO. SIZE LFbpDL5forward 5′-GTC ATT TGG TAG GAGTGG CTG-3′ 6 2,970 bp LFbpDL6reverse 5′-TGG TTG ATA TAG CAC TTG GTGAC-3′ 7 2,970 bp LFbpDL7forward * 5′-GGA TGA CAT GAC AAC TGC TGA TC-3′ 82,639 bp LFbpDL8reverse 5′-CAA TTG TCA GCA CGT CTC TGT AC-3′ 9 2,639 bpLFbpDL9forward 5′-CTT GGA ACT GGT GTT GGT ATG G-3′ 10 2,561 bpLFbpDL10reverse 5′-CAG GTG TTA CTT CAG GTG CTA C-3′ 11 2,561 bp

[0167] 5 PCR reaction was run using an iCycler (BioRad) and conditionsused were:

[0168] Cycle 1: (1X) Step 1: 95° C. for 2 min

[0169] Cycle 2: (30X) Step 1: 94° C. for 30 sec

[0170] Step 2: 94° C. for 30 sec

[0171] Step 3: 68° C. for 3 min

[0172] Cycle 3: (1X) Step 7: 68° C. for 7 min

[0173] Cycle 4: (1X) Step 7: 4° C. holding

[0174] Reactions components used were as follows:

[0175] Primer forward: 0.5 μM

[0176] Primer reverse: 0.5 μM

[0177] Genomic DNA template: 0.5 μg

[0178] dNTP's: 200 μM each

[0179] MgCl₂: 1.5 mM

[0180] Taq polymerase: 0.825 U

[0181] PCR fragments obtained corresponded to the expected theoreticalproduct size (Table 1). These results indicate that the PCR fragmentsobtained show a high degree of similarity with the theoretical SUAMgene. Further confirmation was done to compare the restriction enzymemap of the PCR fragments with the corresponding theoretical SUAMsequence.

[0182] Restriction Digest Confirmation: The longest PCR product of 2,970bp, which includes a start and stop codon and therefore represents theentire gene, was further processed to confirm the specificity of the PCRreaction and further characterize the S. uberis SUAM gene. Restrictionenzyme digestion cuts DNA at specific locations that are recognized bythe different enzymes based upon their nucleotide sequence. The entiregene sequence was analyzed using NEBcutter at the New England BioLabsweb site. Three restriction enzymes, Bcl I, Hpa I, and Nla III werechosen based on their ability to recognize specific sequence sites thatwhen cut would generate distinctly identifiable fragments. TABLE 2Restriction enzymes, site of digestion (coordinates) and expected lengthof digested DNA. Enzyme Coordinates (bp #) Length (bp) Bcl I  329-26322304 Bcl I 2633-3041 409 Bcl I  1-328 328 Hpa I 1625-3041 1417 Hpa I  1-1204 1204 Hpa I 1205-1624 420 Nla III 1580-3041 1462 Nla III 320-1367 1048 Nla III  1-319 319 Nla III 1368-1579 212

[0183] Digestion of the 2,970 bp PCR fragment generated the expectedpatterns (lower molecular weight products were not clearly detected dueto detection limits, as would be expected). The combined results of sixprimer binding sites and 10 restriction cut sites by 3 enzymes confirmedthat PCR fragments have a restriction pattern similar to that of thetheoretical SUAM sequence (FIG. 3, Seq. ID No. 1). These results,together with those from PCR reactions using several primercombinations, indicate that the PCR generated DNA fragment is similar tothe theoretical SUAM nucleic acid sequence.

EXAMPLE 15

[0184] DNA Sequencing of SUAM

[0185] The SUAM gene was amplified, cloned and sequenced from themastitis pathogen S. uberis strain UT 888. The results of thissequencing were that S. uberis SUAM has 99% sequence identity to thetheoretical SUAM gene identified in the Sanger S. uberis genomicdatabase by homology to the reverse translated peptide sequencedescribed in Example 14.

[0186] The 2,970 bp PCR amplicon encompassing the SUAM gene wasgenerated with primers LFbpDL5forward and LFbpDL6reverse shown in Table1 in Example 14 (Seq ID Nos. 6 & 7). The product was gel purified from a1.2% SeaPlaque GTG agarose gel (BioWhittaker Molecular Applications,Rockland, Me.) with the QIAEX II gel extraction kit (Qiagen Inc.,Valencia, Calif.). The cloning into plasmid pCR-XL-TOPO of the purifiedamplicon was by the interaction of nontemplate-dependent polymerasegenerated adenine (A), overhangs of the amplicon and thymine (T), andoverhangs of the vector. A mixture of recombinant Taq polymerase andPyrococus DNA polymerase was used to minimize polymerase reading error(Invitrogen, Carlsbad, Calif.). Chemically competent Escherichia coli,TOP 10 cells, were transformed and selected on Luria-Bertani agar with50 μg/ml kanamycin (Invitrogen, Carlsbad, Calif.). The positive clonewas confirmed by isolation of the plasmid, (Wizard Plus SV miniprep DNApurification system; Promega, Madison, Wis.), re-amplification of theinsert, and digestion with restriction enzymes (New England BioLabs,Inc., Beverly, Mass.) based upon restriction sites picked from thetheoretical sequence.

[0187] Confirmation of the theoretical sequence (The Wellcome TrustSanger Institute, Hinxton, Cambs, UK) and determination of the actualsequence from S. uberis 888 was accomplished by automated DNA sequencing(Molecular Biology Resource Facility, The University of Tennessee,Knoxville, Tenn.) of the plasmid in the region of insertion in both aforward and reverse direction to sequence both strands. The firstprimers were of known sites on the plasmid; M13 forward and M13 reverse,with subsequent primers (Integrated DNA Technologies, Coralville, Ia.)being chosen from the 3′ end of the determined nucleic acid code. Fourrounds of sequencing yielded enough DNA sequence code to transverse theinsert in each direction. Sequence contig assembly was performed withthe aid of the software Sequencher ver. 4.0.2 (Gene Codes Corporation,Ann Arbor, Mich.).

[0188] As each forward and reverse contig was assembled, the overlappingregions provided a quality control check for sequencing error. When theforward and the reverse assembled contigs were compared, this providedan additional quality control check. There were at least two and oftenmore sequencing reactions used for each position in the final nucleicacid sequence. Final comparison and confirmation of the theoreticaldatabase sequence, and actual S. uberis UT 888 sequence were made withBLAST 2 SEQUENCES, BLASTN ver. 2.2.5 (National Center for BiotechnologyInformation, Bethesda, Md.). Results of this alignment were:Identities=2948 (theoretical)/2970 (actual) or 99% similarity.

[0189] The complete SUAM DNA sequence is presented in FIG. 5 and isdesignated Seq. ID No. 5. The complete SUAM gene DNA sequence did notshow homology with other S. uberis genes reported in the Sanger S.uberis genomic database. This indicates that the SUAM gene codes for aunique S. uberis protein.

[0190] The amino acid sequence encoded by the SUAM DNA sequence of Seq.ID No. 5 is presented in FIG. 6. Polypeptide fragments encoded by theDNA Sequence of Seq. ID No. 5 are shown in Seq. ID Nos. 13 to 17,respectively, in order of appearance in FIG. 6. In FIG. 6, the presenceof three sequential asterisks (***) indicates the position of a stopcodon in the nucleotide sequence of Seq. ID No. 5. The underlinedportion of amino acid sequence of FIG. 6 represents the N-terminalsequence of the SUAM protein. The underlined and bold portion of thesequence of FIG. 6 represents pepSUAM.

[0191] The SUAM polypeptide is shown in Seq. ID No. 15, preferably fromamino acids 64 to 905 and most preferably from amino acids 66 to 905.The pepSUAM polypeptide is shown in Seq. ID No. 15 at amino acids 66 to80.

[0192] The terms and expressions which have been employed in theforegoing specification are used as terms of description and notlimitation, and there is no intention that the use of such terms andexpressions excludes equivalents of the features shown and describedabove. Further modifications, uses, and applications of the inventiondescribed herein will be apparent to those skilled in the art. It isintended that such modifications be encompassed in the following claims.

1 17 1 3041 DNA Streptococcus uberis 1 atgattagtc ttctatccga atttgatagtcatttggtag gagtggctgt ttttgctgaa 60 aatgctaaag aagaacgtga acagatggcatataaatcat tgcttaaagt ttctgaaata 120 gatgtcaaga acaataaagt cgtcgttgaagttgggaata tttttaacga tatataatgt 180 atggagagaa aaagggaata ttatggaattcgaaaacaca aaatctaatc agattaaaac 240 aacacttgct ttaacgtcaa cactcgcacttcttggaact ggtgttggta tgggacatac 300 cgttaatgcg gatgacatga caactgctgatcaatcacct aaattacaag gtgaagaagc 360 aacattggcg cctacaaaca ttgaagatactaaagcagcc attgatatta aaacagctac 420 attagcagaa caaaccgatg ctcttaatactgtaaatgag acaatcacaa gcacaaatga 480 agaattagct actttagaag gaggcttagctgataaagaa acagcagttg cagatgctga 540 aaaaacattg gagtctgttt caaatgcctcagaagaagaa tttaatcaat tagcagaaca 600 aaataaagct gacttagcta aaactcaagaggagctaaaa cttgctgaag caacaaaaga 660 agaagttgca acacaggtat tgacacaatctgacgaggta acagctgcag ctaatgaagc 720 taaaaaaatg gctgaaaaag ttgcacaagcagagacaaaa gtttcagact tgacgaaaat 780 ggtcaatcaa ccagaagcaa taacagctcaagttgaaata gaacaaaaca atgtcaaaat 840 catttcggaa gatttagcaa aagccaaaactgatttagtt gctgtaacag ataatacaaa 900 aacacaatta gcaaatgatt tagcgactgctcaatctagc ttaagtgcca aacaaaatga 960 attagctaaa gtacagtcac aaacaagtaatgtcgcagtg aatgttatgg gtgctaataa 1020 aatggttgct ccaactaatt acccaattaatgaaatcaaa aaattaatgt caagtggtta 1080 cattgggaca caatcttatc taaatacattctatgcttta aaagatcaac tggtttctaa 1140 agcagaagtt ggggcatact taaatcattacgttgatatc gcaagtgact taaaccgtat 1200 cgttaaccca gataacttat cagttgaggttcaaaatgaa ttggctgtat ttgcagcaac 1260 attgattaat tctgttcgtc aacaatttggtctttctgca gtcgaagtga cgcaaggtgc 1320 tcaagagttt gctcgcactt tgactcgaaactataaagta acacatggaa acactgttcc 1380 tttctttaat tacaatcaac ctggcaagaatggtcatata ggcattggtc cacacgatag 1440 aacaattatc gaacaagcag ctacaagtgttggcttaaaa gctaatgatg ataatatgta 1500 tgaaaacatc ggattctttg atgatgttcatactgttaat ggtatcaaac gtagtattta 1560 taacagtatt aagtacatgc tgtttacagacttcacctat ggaaatacat ttggacatac 1620 ggttaacttg ttgcgttctg ataaaacaaacccaagtgct ccggtctatt taggagtttc 1680 aacagaaact gttggtggtt taaatacccactatgttatc ttcccggcaa gcaatattgt 1740 aaatgccagc caattcagca aacaagtggtttcaggtcca ttaacaacag ttgataacag 1800 tgctaaaatt agcactcttc aagcaagtattacttctgtt gagtctaaaa ttcaaacctt 1860 acaaaaacgt attgcaaata tttcttcagaagcactagtt gtctctgcac agagaaaagt 1920 agatggttta gctgcaaaac ttcaaaaagctgaatctaac gttgaaaaag caaaagctca 1980 acttcaacag ttacaagatt caaaagaagatttacataaa caacttgctt tttccctttc 2040 aactcgtaag gatttaaaag gtcaacttgacgaatcgctt gttcacctaa atcagtctaa 2100 aattctttta catagcttag aagaaaaacaaagtcaagtg gcaagtcaaa ttaacgtctt 2160 gacattgaag aaggcacaac ttgaaaaagaactagccttt aactctcatc caaatcgtga 2220 aaaagttgca aaagaaaaag ttgaagaggctcaaaaagca ttaacagaaa ccttatctca 2280 aattaaaact aaaaaagcta tcttaaatgatttaacacaa gaaaaagcaa aattgacgtc 2340 agcaatcaca acaactgaac aacaaattgttttgttgaag aatcatttag caaatcaagt 2400 ggcgaatgct ccaaaaatca gcagtattgtccaaagatca gaaaacaata gagtaagacc 2460 tgatgtttct gatacaagag agaaggcagtagatactgct caagaagcga caattcttgc 2520 tcaagcagaa acaatggctg aagaagtcattacaaattct gcaaaagcca ttgttgcaaa 2580 tgctcaaaat gttgcacaag agattatgaaagtagcacct gaagtaacac ctgatcaagg 2640 agttgttgca aaagttgcag ataatattaagaaaaataat gccccagcaa gtaaatcata 2700 tggtgcaagt tcatcaacgg taggaaatgctacttcttca cgagatgaaa gtacaaaacg 2760 tgctttaaga gcaggaattg ttatgctggcagcagcagga cttactggtt acaaactcag 2820 aagagatggc aaaaaataag aaaatcaaaggaaaaattga ttgacagaaa gtaccgtcta 2880 tgttactata gtagacggta ctttttacttttggtctctc aaaagtgtac agagacgtgc 2940 tgacaattgt tgcaaaagta cacacagatataggctgtca ccaagtgcta tatcaaccaa 3000 aaataaaaaa atacaggaga atgtagatgcctacaattaa c 3041 2 1007 PRT Streptococcus uberis 2 Leu Val Phe Tyr ProAsn Leu Ile Val Ile Trp Glu Trp Leu Phe Leu 1 5 10 15 Leu Lys Met LeuLys Lys Asn Val Asn Arg Trp His Ile Asn His Cys 20 25 30 Leu Lys Phe LeuLys Met Ser Arg Thr Ile Lys Ser Ser Leu Lys Leu 35 40 45 Gly Ile Phe LeuThr Ile Tyr Asn Val Trp Arg Glu Lys Gly Asn Ile 50 55 60 Met Glu Phe GluAsn Thr Lys Ser Asn Gln Ile Lys Thr Thr Leu Ala 65 70 75 80 Leu Thr SerThr Leu Ala Leu Leu Gly Thr Gly Val Gly Met Gly His 85 90 95 Thr Val AsnAla Asp Asp Met Thr Thr Ala Asp Gln Ser Pro Lys Leu 100 105 110 Gln GlyGlu Glu Ala Thr Leu Ala Pro Thr Asn Ile Glu Asp Thr Lys 115 120 125 AlaAla Ile Asp Ile Lys Thr Ala Thr Leu Ala Glu Gln Thr Asp Ala 130 135 140Leu Asn Thr Val Asn Glu Thr Ile Thr Ser Thr Asn Glu Glu Leu Ala 145 150155 160 Thr Leu Glu Gly Gly Leu Ala Asp Lys Glu Thr Ala Val Ala Asp Ala165 170 175 Glu Lys Thr Leu Glu Ser Val Ser Asn Ala Ser Glu Glu Glu PheAsn 180 185 190 Gln Leu Ala Glu Gln Asn Lys Ala Asp Leu Ala Lys Thr GlnGlu Glu 195 200 205 Leu Lys Leu Ala Glu Ala Thr Lys Glu Glu Val Ala ThrGln Val Leu 210 215 220 Thr Gln Ser Asp Glu Val Thr Ala Ala Ala Asn GluAla Lys Lys Met 225 230 235 240 Ala Glu Lys Val Ala Gln Ala Glu Thr LysVal Ser Asp Leu Thr Lys 245 250 255 Met Val Asn Gln Pro Glu Ala Ile ThrAla Gln Val Glu Ile Glu Gln 260 265 270 Asn Asn Val Lys Ile Ile Ser GluAsp Leu Ala Lys Ala Lys Thr Asp 275 280 285 Leu Val Ala Val Thr Asp AsnThr Lys Thr Gln Leu Ala Asn Asp Leu 290 295 300 Ala Thr Ala Gln Ser SerLeu Ser Ala Lys Gln Asn Glu Leu Ala Lys 305 310 315 320 Val Gln Ser GlnThr Ser Asn Val Ala Val Asn Val Met Gly Ala Asn 325 330 335 Lys Met ValAla Pro Thr Asn Tyr Pro Ile Asn Glu Ile Lys Lys Leu 340 345 350 Met SerSer Gly Tyr Ile Gly Thr Gln Ser Tyr Leu Asn Thr Phe Tyr 355 360 365 AlaLeu Lys Asp Gln Leu Val Ser Lys Ala Glu Val Gly Ala Tyr Leu 370 375 380Asn His Tyr Val Asp Ile Ala Ser Asp Leu Asn Arg Ile Val Asn Pro 385 390395 400 Asp Asn Leu Ser Val Glu Val Gln Asn Glu Leu Ala Val Phe Ala Ala405 410 415 Thr Leu Ile Asn Ser Val Arg Gln Gln Phe Gly Leu Ser Ala ValGlu 420 425 430 Val Thr Gln Gly Ala Gln Glu Phe Ala Arg Thr Leu Thr ArgAsn Tyr 435 440 445 Lys Val Thr His Gly Asn Thr Val Pro Phe Phe Asn TyrAsn Gln Pro 450 455 460 Gly Lys Asn Gly His Ile Gly Ile Gly Pro His AspArg Thr Ile Ile 465 470 475 480 Glu Gln Ala Ala Thr Ser Val Gly Leu LysAla Asn Asp Asp Asn Met 485 490 495 Tyr Glu Asn Ile Gly Phe Phe Asp AspVal His Thr Val Asn Gly Ile 500 505 510 Lys Arg Ser Ile Tyr Asn Ser IleLys Tyr Met Leu Phe Thr Asp Phe 515 520 525 Thr Tyr Gly Asn Thr Phe GlyHis Thr Val Asn Leu Leu Arg Ser Asp 530 535 540 Lys Thr Asn Pro Ser AlaPro Val Tyr Leu Gly Val Ser Thr Glu Thr 545 550 555 560 Val Gly Gly LeuAsn Thr His Tyr Val Ile Phe Pro Ala Ser Asn Ile 565 570 575 Val Asn AlaSer Gln Phe Ser Lys Gln Val Val Ser Gly Pro Leu Thr 580 585 590 Thr ValAsp Asn Ser Ala Lys Ile Ser Thr Leu Gln Ala Ser Ile Thr 595 600 605 SerVal Glu Ser Lys Ile Gln Thr Leu Gln Lys Arg Ile Ala Asn Ile 610 615 620Ser Ser Glu Ala Leu Val Val Ser Ala Gln Arg Lys Val Asp Gly Leu 625 630635 640 Ala Ala Lys Leu Gln Lys Ala Glu Ser Asn Val Glu Lys Ala Lys Ala645 650 655 Gln Leu Gln Gln Leu Gln Asp Ser Lys Glu Asp Leu His Lys GlnLeu 660 665 670 Ala Phe Ser Leu Ser Thr Arg Lys Asp Leu Lys Gly Gln LeuAsp Glu 675 680 685 Ser Leu Val His Leu Asn Gln Ser Lys Ile Leu Leu HisSer Leu Glu 690 695 700 Glu Lys Gln Ser Gln Val Ala Ser Gln Ile Asn ValLeu Thr Leu Lys 705 710 715 720 Lys Ala Gln Leu Glu Lys Glu Leu Ala PheAsn Ser His Pro Asn Arg 725 730 735 Glu Lys Val Ala Lys Glu Lys Val GluGlu Ala Gln Lys Ala Leu Thr 740 745 750 Glu Thr Leu Ser Gln Ile Lys ThrLys Lys Ala Ile Leu Asn Asp Leu 755 760 765 Thr Gln Glu Lys Ala Lys LeuThr Ser Ala Ile Thr Thr Thr Glu Gln 770 775 780 Gln Ile Val Leu Leu LysAsn His Leu Ala Asn Gln Val Ala Asn Ala 785 790 795 800 Pro Lys Ile SerSer Ile Val Gln Arg Ser Glu Asn Asn Arg Val Arg 805 810 815 Pro Asp ValSer Asp Thr Arg Glu Lys Ala Val Asp Thr Ala Gln Glu 820 825 830 Ala ThrIle Leu Ala Gln Ala Glu Thr Met Ala Glu Glu Val Ile Thr 835 840 845 AsnSer Ala Lys Ala Ile Val Ala Asn Ala Gln Asn Val Ala Gln Glu 850 855 860Ile Met Lys Val Ala Pro Glu Val Thr Pro Asp Gln Gly Val Val Ala 865 870875 880 Lys Val Ala Asp Asn Ile Lys Lys Asn Asn Ala Pro Ala Ser Lys Ser885 890 895 Tyr Gly Ala Ser Ser Ser Thr Val Gly Asn Ala Thr Ser Ser ArgAsp 900 905 910 Glu Ser Thr Lys Arg Ala Leu Arg Ala Gly Ile Val Met LeuAla Ala 915 920 925 Ala Gly Leu Thr Gly Tyr Lys Leu Arg Arg Asp Gly LysLys Glu Asn 930 935 940 Gln Arg Lys Asn Leu Thr Glu Ser Thr Val Tyr ValThr Ile Val Asp 945 950 955 960 Gly Thr Phe Tyr Phe Trp Ser Leu Lys SerVal Gln Arg Arg Ala Asp 965 970 975 Asn Cys Cys Lys Ser Thr His Arg TyrArg Leu Ser Pro Ser Ala Ile 980 985 990 Ser Thr Lys Asn Lys Lys Ile GlnGlu Asn Val Asp Ala Tyr Asn 995 1000 1005 3 2971 DNA Streptococcusuberis 3 gtcatttggt aggagtggct gtttttgctg aaaatgctaa agaagaacgtgaacagatgg 60 catataaatc attgcttaaa gtttctgaaa tagatgtcaa gaacaataaagtcgtcgttg 120 aagttgggaa tatttttaac gatatataat gtatggagag aaaaagggaatattatggaa 180 ctcgaaaaca caaaatctaa tcagattaaa acaacacttg ctttaacgtcaacactcgca 240 cttcttggaa ctggtgttgg tatgggacat accgttaatg cggatgacatgacaactgct 300 gatcaatcac ctaaattaca aggtgaagaa gcaacattgg cgcctacaaacattgaagat 360 actaaagcag ccattgatac taaaacagct acattagcag aacaaaccgatgctcttaat 420 actgtaaatg agacaatcac aagcacaaat gaagaattag ctactttagaaggaggctta 480 gctgataaag aaacagcagt tgcagatgct gaaaaaacat tggagtctgtttcaaatgcc 540 tcagaagaag aatttaatca attagcagaa caaaataaag ctgacttagctaaaactcaa 600 gaggagctaa aacttgctga agcaacaaaa gaagaagttg caacacaggtattgacacaa 660 tctgacgagg taacagctgc agctaatgaa gctaaaaaaa tggctgaaaaagttgcacaa 720 gcagagacaa aagtttcaga cttgacgaaa atggtcaatc aaccagaagcaataacagct 780 caagttgaaa tagaacaaaa caatgtcaaa atcatttcgg aagatttagcaaaagccaaa 840 actgatttag ttgctgtaac agataataca aaaacacaat tagcaaatgatttagcgact 900 gctcaatcta gcttaagtgc caaacaaaat gaattagcta aagtacagtcacaaacaagt 960 aatgtcgcag tgaatgttat gggtgctaat aaaatggttg ctccaactaattacccaatt 1020 aatgaaatca aaaaattaat gtcaagtggt tacattggga cacaatcttatctaaataca 1080 ttctatgctt taaaagatca actggtttct aaagcagaag ttggggcatacttaaatcat 1140 tacgttgata tcgcaagtga cttaaaccgt atcgttaacc cagataacttatcagttgag 1200 gttcaaaatg aattggctgt atttgcagca acattgatta attctgttcgtcagcaattt 1260 ggtctttctg cagtcgaagt gacgcaaggt gctcaagagt ttgctcgcactttgactcaa 1320 aactataaag caacacatgg aaacactgtt cctttcttta attacaatcaacctggcaag 1380 aatggtcata taggcattgg tccacacgat agaacaatta tcgaacaagcagctacaagt 1440 gttggcttaa aagctaatga tgataacatg tatgaaaaca tcggattctttgatgatgtt 1500 catactgtta atggtatcaa acgtagtatt tataacagta ttaagtacatgctgtttaca 1560 gacctcacct atggaaatac atttggacat acggttaact tgttgcgttctgataaaaca 1620 aacccaagtg ctccggtcta tttaggagtt tcaacagaaa ctgttggtggtttaaatacc 1680 cactatgtta tcttcccggc aagcaatatt gtaaatgcca gccagttcagcaaacaagtg 1740 gtttcaggtc cattaacaac agttgataac agtgctaaaa ttagcactcttcaagcaagt 1800 attgcttctg ttgagtctaa aattcaaacc ttacaaaaac gtattgcaaatatttcttca 1860 gaagcactag ttatctctgc acagagaaaa gtagatggtt tagctgcaaaacttcaaaaa 1920 gctgaatcta acgttgaaaa agcaaaagct caacttcaac agttaaaagattcaaaagaa 1980 gatttacata aacaacttgc ttttgccctt tcaactcgta aggatttaaaaggtcaactt 2040 gacgaatcgc ttgttcacct aaatcagtct aaaattcttt ttcatagcttagaagaaaaa 2100 caaagtcaag tggcaagtca aattaacgtc ttgacattga agaaggcacaacttgaaaaa 2160 gaactagcct ttaactctca tccaaatcgt gaaaaagttg caaaagaaaaagttgaagag 2220 gctcaaaaag cattaacaga aaccttatct caaattaaaa ctaaaaaagctatcttaaat 2280 gatttaacac aagaaaaagc aaaattgacg tcagcaatca caacaactgaacaacaaatt 2340 gttttgttga agaatcattt agcaaatcaa gtggcgaatg ctccaaaaatcagcagtatt 2400 gtccaaagat cagaaaacaa tggagtaaga cctgatgttt ctgatacaagagagaaggca 2460 gtagatactg ctcaagaagc gacaattctt gctcaagcag aaacaatggctgaagaagtc 2520 attacaaatt ctgcaaaagc cattgttgca aatgctcaaa atgttgcacaagagattatg 2580 aaagtagcac ctgaagtaac acctgatcaa ggagttgttg caaaagttgcagataatatt 2640 aagaaaaata atgccccagc aagtaaatca tatggtgcaa gttcatcaactgtaggaaat 2700 gctacttctt cacgagatga aagtacaaaa cgtgctttaa gagcaggaattgttatgctg 2760 gcagcagcag gacttactgg ttacaaactc agaagagatg gcaaaaaataagaaaatcaa 2820 aggaaaaatt gattgacaga aagtaccgtc tatgttacta tagtagacggtactttttac 2880 ttttggtctc tcaaaagtgt acagagacgt gctgacaatt gttgcaaaagtacacacaga 2940 tataggctgt caccaagtgc tatatcaacc a 2971 4 15 PRTStreptococcus uberis 4 Met Thr Thr Ala Asp Gln Ser Pro Lys Leu Gln GlyGlu Glu Ala 1 5 10 15 5 45 DNA Streptococcus uberis 5 atgacaactgctgatcaatc acctaaatta caaggtgaag aagca 45 6 21 DNA Artificial SequenceDescription of Artificial Sequence Primer 6 gtcatttggt aggagtggct g 21 723 DNA Artificial Sequence Description of Artificial Sequence Primer 7tggttgatat agcacttggt gac 23 8 23 DNA Artificial Sequence Description ofArtificial Sequence Primer 8 ggatgacatg acaactgctg atc 23 9 23 DNAArtificial Sequence Description of Artificial Sequence Primer 9caattgtcag cacgtctctg tac 23 10 22 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 10 cttggaactg gtgttggtat gg 22 11 22 DNAArtificial Sequence Description of Artificial Sequence Primer 11caggtgttac ttcaggtgct ac 22 12 20 PRT Streptococcus uberis MOD_RES (17)Thr or Ala 12 Asp Met Thr Thr Ala Asp Gln Ser Pro Lys Leu Gln Gly GluGlu Ala 1 5 10 15 Xaa Leu Xaa Xaa 20 13 3 PRT Streptococcus uberis 13Val Ile Trp 1 14 26 PRT Streptococcus uberis 14 Glu Trp Leu Phe Leu LeuLys Met Leu Lys Lys Asn Val Asn Arg Trp 1 5 10 15 His Ile Asn His CysLeu Lys Phe Leu Lys 20 25 15 905 PRT Streptococcus uberis 15 Met Ser ArgThr Ile Lys Ser Ser Leu Lys Leu Gly Ile Phe Leu Thr 1 5 10 15 Ile TyrAsn Val Trp Arg Glu Lys Gly Asn Ile Met Glu Leu Glu Asn 20 25 30 Thr LysSer Asn Gln Ile Lys Thr Thr Leu Ala Leu Thr Ser Thr Leu 35 40 45 Ala LeuLeu Gly Thr Gly Val Gly Met Gly His Thr Val Asn Ala Asp 50 55 60 Asp MetThr Thr Ala Asp Gln Ser Pro Lys Leu Gln Gly Glu Glu Ala 65 70 75 80 ThrLeu Ala Pro Thr Asn Ile Glu Asp Thr Lys Ala Ala Ile Asp Thr 85 90 95 LysThr Ala Thr Leu Ala Glu Gln Thr Asp Ala Leu Asn Thr Val Asn 100 105 110Glu Thr Ile Thr Ser Thr Asn Glu Glu Leu Ala Thr Leu Glu Gly Gly 115 120125 Leu Ala Asp Lys Glu Thr Ala Val Ala Asp Ala Glu Lys Thr Leu Glu 130135 140 Ser Val Ser Asn Ala Ser Glu Glu Glu Phe Asn Gln Leu Ala Glu Gln145 150 155 160 Asn Lys Ala Asp Leu Ala Lys Thr Gln Glu Glu Leu Lys LeuAla Glu 165 170 175 Ala Thr Lys Glu Glu Val Ala Thr Gln Val Leu Thr GlnSer Asp Glu 180 185 190 Val Thr Ala Ala Ala Asn Glu Ala Lys Lys Met AlaGlu Lys Val Ala 195 200 205 Gln Ala Glu Thr Lys Val Ser Asp Leu Thr LysMet Val Asn Gln Pro 210 215 220 Glu Ala Ile Thr Ala Gln Val Glu Ile GluGln Asn Asn Val Lys Ile 225 230 235 240 Ile Ser Glu Asp Leu Ala Lys AlaLys Thr Asp Leu Val Ala Val Thr 245 250 255 Asp Asn Thr Lys Thr Gln LeuAla Asn Asp Leu Ala Thr Ala Gln Ser 260 265 270 Ser Leu Ser Ala Lys GlnAsn Glu Leu Ala Lys Val Gln Ser Gln Thr 275 280 285 Ser Asn Val Ala ValAsn Val Met Gly Ala Asn Lys Met Val Ala Pro 290 295 300 Thr Asn Tyr ProIle Asn Glu Ile Lys Lys Leu Met Ser Ser Gly Tyr 305 310 315 320 Ile GlyThr Gln Ser Tyr Leu Asn Thr Phe Tyr Ala Leu Lys Asp Gln 325 330 335 LeuVal Ser Lys Ala Glu Val Gly Ala Tyr Leu Asn His Tyr Val Asp 340 345 350Ile Ala Ser Asp Leu Asn Arg Ile Val Asn Pro Asp Asn Leu Ser Val 355 360365 Glu Val Gln Asn Glu Leu Ala Val Phe Ala Ala Thr Leu Ile Asn Ser 370375 380 Val Arg Gln Gln Phe Gly Leu Ser Ala Val Glu Val Thr Gln Gly Ala385 390 395 400 Gln Glu Phe Ala Arg Thr Leu Thr Gln Asn Tyr Lys Ala ThrHis Gly 405 410 415 Asn Thr Val Pro Phe Phe Asn Tyr Asn Gln Pro Gly LysAsn Gly His 420 425 430 Ile Gly Ile Gly Pro His Asp Arg Thr Ile Ile GluGln Ala Ala Thr 435 440 445 Ser Val Gly Leu Lys Ala Asn Asp Asp Asn MetTyr Glu Asn Ile Gly 450 455 460 Phe Phe Asp Asp Val His Thr Val Asn GlyIle Lys Arg Ser Ile Tyr 465 470 475 480 Asn Ser Ile Lys Tyr Met Leu PheThr Asp Leu Thr Tyr Gly Asn Thr 485 490 495 Phe Gly His Thr Val Asn LeuLeu Arg Ser Asp Lys Thr Asn Pro Ser 500 505 510 Ala Pro Val Tyr Leu GlyVal Ser Thr Glu Thr Val Gly Gly Leu Asn 515 520 525 Thr His Tyr Val IlePhe Pro Ala Ser Asn Ile Val Asn Ala Ser Gln 530 535 540 Phe Ser Lys GlnVal Val Ser Gly Pro Leu Thr Thr Val Asp Asn Ser 545 550 555 560 Ala LysIle Ser Thr Leu Gln Ala Ser Ile Ala Ser Val Glu Ser Lys 565 570 575 IleGln Thr Leu Gln Lys Arg Ile Ala Asn Ile Ser Ser Glu Ala Leu 580 585 590Val Ile Ser Ala Gln Arg Lys Val Asp Gly Leu Ala Ala Lys Leu Gln 595 600605 Lys Ala Glu Ser Asn Val Glu Lys Ala Lys Ala Gln Leu Gln Gln Leu 610615 620 Lys Asp Ser Lys Glu Asp Leu His Lys Gln Leu Ala Phe Ala Leu Ser625 630 635 640 Thr Arg Lys Asp Leu Lys Gly Gln Leu Asp Glu Ser Leu ValHis Leu 645 650 655 Asn Gln Ser Lys Ile Leu Phe His Ser Leu Glu Glu LysGln Ser Gln 660 665 670 Val Ala Ser Gln Ile Asn Val Leu Thr Leu Lys LysAla Gln Leu Glu 675 680 685 Lys Glu Leu Ala Phe Asn Ser His Pro Asn ArgGlu Lys Val Ala Lys 690 695 700 Glu Lys Val Glu Glu Ala Gln Lys Ala LeuThr Glu Thr Leu Ser Gln 705 710 715 720 Ile Lys Thr Lys Lys Ala Ile LeuAsn Asp Leu Thr Gln Glu Lys Ala 725 730 735 Lys Leu Thr Ser Ala Ile ThrThr Thr Glu Gln Gln Ile Val Leu Leu 740 745 750 Lys Asn His Leu Ala AsnGln Val Ala Asn Ala Pro Lys Ile Ser Ser 755 760 765 Ile Val Gln Arg SerGlu Asn Asn Gly Val Arg Pro Asp Val Ser Asp 770 775 780 Thr Arg Glu LysAla Val Asp Thr Ala Gln Glu Ala Thr Ile Leu Ala 785 790 795 800 Gln AlaGlu Thr Met Ala Glu Glu Val Ile Thr Asn Ser Ala Lys Ala 805 810 815 IleVal Ala Asn Ala Gln Asn Val Ala Gln Glu Ile Met Lys Val Ala 820 825 830Pro Glu Val Thr Pro Asp Gln Gly Val Val Ala Lys Val Ala Asp Asn 835 840845 Ile Lys Lys Asn Asn Ala Pro Ala Ser Lys Ser Tyr Gly Ala Ser Ser 850855 860 Ser Thr Val Gly Asn Ala Thr Ser Ser Arg Asp Glu Ser Thr Lys Arg865 870 875 880 Ala Leu Arg Ala Gly Ile Val Met Leu Ala Ala Ala Gly LeuThr Gly 885 890 895 Tyr Lys Leu Arg Arg Asp Gly Lys Lys 900 905 16 6 PRTStreptococcus uberis 16 Glu Asn Gln Arg Lys Asn 1 5 17 46 PRTStreptococcus uberis 17 Leu Thr Glu Ser Thr Val Tyr Val Thr Ile Val AspGly Thr Phe Tyr 1 5 10 15 Phe Trp Ser Leu Lys Ser Val Gln Arg Arg AlaAsp Asn Cys Cys Lys 20 25 30 Ser Thr His Arg Tyr Arg Leu Ser Pro Ser AlaIle Ser Thr 35 40 45

1. An isolated polypeptide comprising the amino acid sequence of Seq. IDNo. 4 or an amino acid sequence that is at least 50% homologous withSeq. ID No. 4, wherein an antibody that binds to the polypeptideinhibits the adherence or internalization of Streptococcus uberis tobovine mammary cells.
 2. The isolated polypeptide of claim 1 which is atleast 60% homologous with Seq. ID No.
 4. 3. The isolated polypeptide ofclaim 1 which is at least 70% homologous with Seq. ID No.
 4. 4. Theisolated polypeptide of claim 1 which is at least 80% homologous withSeq. ID No.
 4. 5. The isolated polypeptide of claim 1 which is at least90% homologous with Seq. ID No.
 4. 6. An isolated polypeptide comprisingthe amino acid sequence of Seq. ID No. 2 or Seq. ID No. 15, or an aminoacid sequence that is at least 50% homologous with either Seq. ID No. 2or Seq. ID No. 15, wherein an antibody that binds to the polypeptideinhibits the adherence or internalization of Streptococcus uberis tobovine mammary cells.
 7. The isolated polypeptide of claim 6 which is atleast 60% homologous with Seq. ID No. 2 or Seq. No.
 15. 8. The isolatedpolypeptide of claim 6 which is at least 70% homologous with Seq. ID No.2 or Seq. No.
 15. 9. The isolated polypeptide of claim 6 which is atleast 80% homologous with Seq. ID No. 2 or Seq. No.
 15. 10. The isolatedpolypeptide of claim 6 which is at least 90% homologous with Seq. ID No.2 of Seq. No.
 15. 11. An isolated polypeptide comprising at least 6sequential amino acids of Seq. ID No. 4, wherein an antibody that bindsto the polypeptide inhibits the adherence or internalization ofStreptococcus uberis to bovine mammary cells.
 12. The polypeptide ofclaim 11 which comprises at least 7 to 10 sequential amino acids of Seq.ID No.
 4. 13. The polypeptide of claim 11 which comprises at least 9 to12 sequential amino acids of Seq. ID No.
 4. 14. The polypeptide of claim11 which comprises at least 10 to 15 sequential amino acids of Seq. IDNo.
 4. 15. An isolated nucleic acid comprising a nucleotide sequencethat hybridizes under highly stringent conditions to the complement ofthe nucleotide sequence of Seq. ID No.
 5. 16. The nucleic acid of claim15 that comprises the nucleotide sequence of Seq. ID No.
 5. 17. Thenucleic acid of claim 15 that encodes a polypeptide comprising the aminoacid sequence of Seq. ID No.
 4. 18. An isolated nucleic acid comprisinga nucleotide sequence that hybridizes under highly stringent conditionsto the complement of the nucleotide sequence of Seq. ID No. 1 or Seq. IDNo.
 3. 19. The nucleic acid of claim 18 that hybridizes under highlystringent conditions to the complement of the nucleotide sequence ofnucleotides 311 to 2836 of Seq. ID No. 1 or of nucleotides 283 to 2808of Seq. ID No.
 3. 20. The nucleic acid of claim 18 that hybridizes underhighly stringent conditions to the complement of the nucleotide sequenceof nucleotides 317 to 2836 of Seq. ID No. 1 or nucleotides 289 to 2808of Seq. ID No.
 3. 21. The nucleic acid of claim 18 that comprises thenucleotide sequence of Seq. ID No.
 1. 22. The nucleic acid of claim 21that comprises the nucleotide sequence of nucleotides 317 to 2836 ofSeq. ID No.
 1. 23. The nucleic acid of claim 22 that comprises thenucleotide sequence of nucleotides 311 to 2836 of Seq. ID No.
 1. 24. Thenucleic acid of claim 18 that comprises the nucleotide sequence of Seq.ID No.
 3. 25. The nucleic acid of claim 24 that comprises the nucleotidesequence of nucleotides 289 to 2808 of Seq. ID No.
 3. 26. The nucleicacid of claim 25 that comprises the nucleotide sequence of nucleotides283 to 2808 of Seq. ID No.
 3. 27. An isolated polypeptide that isencoded by the nucleic acid of claim
 18. 28. An isolated polypeptidethat is encoded by the nucleic acid of claim
 19. 29. An isolatedpolypeptide that is encoded by the nucleic acid of claim
 20. 30. Anisolated polypeptide that is encoded by the nucleic acid of claim 21.31. An isolated polypeptide that is encoded by the nucleic acid of claim22.
 32. An isolated polypeptide that is encoded by the nucleic acid ofclaim
 23. 33. An isolated polypeptide that is encoded by the nucleicacid of claim
 24. 34. An isolated polypeptide that is encoded by thenucleic acid of claim
 25. 35. An isolated polypeptide that is encoded bythe nucleic acid of claim
 26. 36. An isolated antibody that selectivelybinds to a polypeptide having an amino acid sequence of any 6 to 15sequential amino acids of Seq. ID No.
 4. 37. The antibody of claim 36which inhibits the adherence or the internalization of S. uberis tobovine mammary epithelial cells.
 38. The antibody of claim 37 which is amonoclonal antibody.
 39. The antibody of claim 37 which is a polyclonalantibody.
 40. An isolated antibody that selectively binds to apolypeptide that is encoded by the nucleic acid of Seq. ID No. 1 or 3 orto a polypeptide that is encoded by a nucleic acid that hybridizes underhighly stringent conditions to the complement of the nucleotide sequenceof Seq. ID No. 1 or Seq. ID No.
 3. 41. The antibody of claim 40 whichinhibits the adherence or the internalization of S. uberis to bovinemammary epithelial cells.
 42. The antibody of claim 40 which is amonoclonal antibody.
 43. The antibody of claim 40 which is a polyclonalantibody.
 44. An isolated nucleic acid comprising a nucleotide sequenceselected from the group consisting of Seq. ID Nos. 6, 7, 8, 9, 10, and11.
 45. The nucleic acid of claim 44 which consists of a nucleotidesequence selected from the group consisting of Seq. ID Nos. 6, 7, 8, 9,10, and 11.